European Society of Sexual Medicine consensus statement on the use of animal models for studying Peyronie’s disease

Abstract Introduction Animal models are frequently used for translational research in Peyronie’s disease (PD). However, due to lack of availability of guidelines, there is some heterogeneity in study design, data reporting, and outcome measures. Aim This European Society for Sexual Medicine consensus statement aims to provide guidance in utilization of animal models in PD research in a standardized and uniform fashion. Methods PubMed was searched for studies using animal models for PD. The following search terms were used: (“Peyronie’s disease” OR “penile fibrosis” OR “penile curvature” OR “induration penis plastica” OR “erectile dysfunction”) AND (“rodent” OR “mouse” OR “mice” OR “rat” OR “rabbit”). Outcomes This European Society for Sexual Medicine statement describes best practice guidelines for utilization of animals in PD research: power calculation, details of available models, surgical procedures, and measurement techniques, while highlighting possible pitfalls and translational limitations of the models. Results In total, 2490 studies were retrieved and 2446 articles were excluded. A total of 44 studies were included, of which 40 studies used rats, 1 study used both rats and mice, 1 study used a genetic mouse model, and 2 studies used rabbits. A significant number of the studies (70.5%) used transforming growth factor β 1 for induction of fibrosis. Oxford 2011 Levels of Evidence criteria could not be applied due to the nature of the studies. Conclusion Despite certain limitations of PD animal models presented, we aimed to provide guidance for their appropriate use in translational research, with the purpose of improving study quality and reproducibility as well as facilitating interpretation of reported results and conclusions.


Introduction
Peyronie's disease (PD) is characterized by localized fibrotic plaque formation in the penile tunica albuginea (TA) 1 with a prevalence of 0.3% to 9%. 2,3 PD presents in 2 phases. First, an acute (unstable) phase, which can present with pain during erection and is characterized by progressive development of the fibrotic plaque. This is followed by a chronic phase that begins to stabilize 12 to 18 months after the first symptoms, along with improvement of pain with a stable penile plaque and curvature. 4 Permanent curvature or plaque induration can have drastic consequences, such as difficulties to penetrate, in some cases severe erectile dysfunction, and can affect the quality of life. [4][5][6] Although the etiology of PD is not clear, repetitive microtrauma to the TA, caused by not fully rigid erection, is generally accepted to be the trigger for inflammation which leads to aberrant wound healing and extracellular matrix (ECM) protein (mostly collagen and elastin) deposition and disorganization. In the chronic phase of PD, this can even lead to calcification of the plaque. 7,8 In addition, a genetic predisposition with other fibrotic disorders such as Dupuytren's disease has been suggested, along with a single nucleotide polymorphism (G915C) in the gene for transforming growth factor beta 1 (TGF-β1). 9,10 Regardless of the triggers, a common feature of all fibrotic diseases, including PD, is the activation of ECM-producing myofibroblasts, which are the key mediators of fibrotic tissue remodeling. 11,12 In an effort to understand the pathophysiology of PD and to develop novel medicines to treat the condition, animal models that resemble the human condition have been developed. Mouse, rat, and rabbit models have been utilized to study PD in vivo. 11, Most models rely on inducing fibrosis in TA either by increasing TGF-β1 in TA or by surgical trauma of the TA. The Tight-skin 1 (Tsk) model is the only genetic model for the disease in which PD-like fibrosis develops spontaneously. 38 Multiple preclinical studies have used PD animal models to investigate the antifibrotic efficacy of various molecules and treatments. 56 Endpoints typically include improved cavernosal smooth muscle/collagen ratios on immunohistochemical staining or messenger RNA and protein content of fibrosisrelated genes and proteins such as α-smooth muscle actin, collagens, and elastin. 57 Moreover, erectile function assessments are often included by measuring intracavernous pressure (ICP) in response to cavernous nerve electrostimulation. 48 However, despite the promising results from preclinical studies, most clinical trials have failed to confirm any structural or lasting benefit of the treatments that were efficacious in animal models in improving the curvature in PD patients, meaning that most of these animal studies failed to translate to the clinical outcomes. [58][59][60] Recently, a preclinical study that utilized TGF-β1-induced PD model in rats 39 showed promising initial results in the clinic. 61,62 Poor design of both clinical and preclinical studies could be partly responsible for this lack of translation. Another cause could be the high variance in methodologies and subsequent reported outcomes when conducting animal studies. A similar discrepancy has been noted in other animal models used in the sexual medicine field, such as the cavernous nerve injury model. This model uses similar techniques and outcome measurements as in PD. 63 These methodological inconsistencies and the lack of consensus guidelines surrounding this topic raise the concern of an increasing number of studies being published with nonreproducible results, leading to little progress in the field.
The aim of this statement paper is to review the current state of art, highlight possible pitfalls, and provide guiding statements for experimental design, technique, and reporting of results when using animal models of PD and to provide further standardization and improvement in the quality of research in this field on behalf of the European Society of Sexual Medicine (ESSM).

Literature search and study eligibility
A literature search of full-text English language publications on basic science studies using an in vivo rodent animal model for PD was performed using PubMed on July 30, 2020, and repeated on February 23, 2023, with the search term ("Peyronie's disease" OR "penile fibrosis" OR "penile curvature" OR "induration penis plastica" OR "erectile dysfunction") AND (rodent OR mouse OR mice OR rat OR rabbit).
Inclusion criteria were peer-reviewed publications with primary data, English language, studies using an in vivo animal model of PD, and studies with the aim to develop or evaluate an animal model for PD or to test a treatment using an animal model for PD.
Exclusion criteria were a secondary data research such as abstracts, letters, and reviews, studies using an in vivo animal model of fibrosis of penile corpus cavernosum only, and studies using an in vivo animal model in which TA grafts are investigated.

Data extraction
The studies were reviewed by 2 different persons (O.O.C., F.C.). If it was not clear from the abstract whether the article might contain relevant data, the full article was assessed.

Review methods
Thereafter, relevant studies were identified, analyzed, and summarized after an interactive peer-review process by the entire panel to obtain a narrative review. The statements were discussed among the panel members. Disagreements were resolved by consensus. It was not possible to grade the studies according to the Oxford 2011 Levels of Evidence criteria 64 because of the nature (basic science) of the studies. The ESSM position is summarized in the specific statements.

General observations
Of 2490 studies retrieved, 2446 articles were excluded. A total of 44 studies were included in this review 11, which are summarized in Table 1. Among them, 40 studies used rats only, 1 study used both rats and mice, 1 study used a genetic mouse model, and 2 studies used rabbits (Table 1). A total of 31 (70.5%) of the 44 studies used either injection of TGF-β1 into TA or increased TGF-β1 levels using viruses or plasmids. The remaining studies used one of the following methods: surgical trauma to the TA, TA injection of fibrin, thrombin, chlorhexidine, allograft of TA tissue, extracorporeal shockwave, human PD cells, or rat blood or plasma (Table 1).
A total of 40 (90.9%) of 44 studies used a negative control in which the vehicle of the substance was injected into the TA or a sham operation was performed (Table 1). A total of 9 (20.5%) studies repeated the fibrosis inducing agents more than once at various time points. A total of 28 (63.6%) studies utilized the PD animal model to test the preventive (n = 21 [47.7%]) and/or therapeutic (n = 16 [36.4%]) effect of an agent (Table 1). A total of 3 (7%) studies reported a power calculation method by which the number of animals was determined.

Statements
Statement #1: We suggest adherence to ARRIVE guidelines.

Evidence and remarks
The ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines are a checklist of information to include in publications describing animal research. Any manuscript that describes animal research is expected to submit a completed ARRIVE checklist, which is now mandated by a majority of publishers. Reporting animal research in adherence with the ARRIVE guidelines ensures transparent and thorough reporting that enables the readers and reviewers to scrutinize the research adequately, evaluate its methodological rigor, and reproduce the methods or findings. 65 A recent study demonstrated that a mandatory ARRIVE checklist during submission is not enough to improve compliance, and more stringent editorial policies are required. 66 Therefore, adherence to the ARRIVE checklist completion does not necessarily mean that the authors complied with each of items in the guidelines. It should also be noted that the ARRIVE guidelines were introduced in 2010. Out of the 44 studies included in this review, 27 were published after 2010. We were not able to verify whether each of the 27 studies submitted a completed ARRIVE checklist. Although the assessment of these studies for their compliance with the ARRIVE guidelines was not within the scope of this review, the ARRIVE guidelines were utilized as steering points in some of the recommendations made subsequently.
Statement #2: We suggest reporting the age and weight of the animals at study entry. Weight of the animals at the endpoint should be also reported.

Evidence
In accordance with the ARRIVE guidelines, 65 the species, strain, age, and weight of the animals should be reported. All of the 44 studies identified in this review have documented the species and the strain. The age and weight were documented in 65.9% and 45.5% of the 44 studies, respectively. Particularly, it was noted that the age of the rats varied greatly from 2 to 11 months (Table 1).

Remarks
The age is particularly pertinent to animal studies in sexual medicine field, as the age is an important determinant of sexual maturity 67 and aging is known to affect sexual behavior and function. 68 We suggest using 10-to 12-week-old Sprague Dawley/CD rats, which are known to reach sexual maturity at around that age. 69 For other species or strains, a literature search should be carried out to find the relevant age when the animal reaches puberty and becomes a sexually mature adult. This information should be included in the paper. It should also be noted that although PD can occur in any age, the prevalence of the condition has been shown to increase with age, 70 hence it is more common in men in their 50s and 60s. If PD is to be studied in older age population, rats that are 18 to 24 months old should be utilized. 67 None of the studies reported the weight of animals at the end of the study. This is particularly important in models in which surgery is involved; any weight loss due to surgeryinduced stress should be documented.

Evidence
The ARRIVE guidelines state that the exact number of animals allocated to each group and the total number of animals in each experiment should be specified. How the sample size was decided should be explained; the details of any sample size calculation should be reported. 65 All 44 studies documented the total number of animals and the number of animals per group. The average number of animals per group was 6.7 ± 2.6 with a wide range of between 3 and 18 ( Table 1). Only 3 (6.8%) studies documented sample size calculations. 19,21,22 Remarks Sample size is crucial to assess the validity of the statistical analysis method and the robustness of the results. Sample size calculations should be performed prior to design of the study using freely available software or algorithms. Consultation with a statistician is also suggested. When reporting sample size calculations, the analysis method, the effect size, the estimate of variability, and the power selected should be documented. It should be remembered that low-powered studies are likely to have more false positives and negatives and would risk wasting animals used in inconclusive research. 71 Statement #4: We suggest using a negative control group.

Evidence
According to the ARRIVE guidelines, a control group should be available. If no control group has been used, the rationale should be stated. 65 Four (9%) of 44 studies did not use a negative control, such as injection of the vehicle substance into TA, and no rationale for the lack of a control group was offered (Table 1).

Remarks
A negative control group would ideally be the vehicle of the substance in which the fibrosis inducer (eg, TGF-β1) is dissolved, such as saline or citrate buffer. If the fibrosis is induced by trauma, allograft, shockwave device, or similar nonpharmacological means, a sham control group should be included. The lack of a negative control group without any rationale is concerning because negative control groups are used to determine whether a difference observed in the intervention group is caused by the intervention, not by chance. Without an appropriate negative control group, it cannot be ascertained whether the fibrosis observed in the study is indeed caused by the inducer.

Evidence
In accordance with the ARRIVE guidelines, for each experimental group, including controls, the authors should describe the procedures in enough detail to allow others to replicate them, including what was done, how it was done, what was used, when and how often, where, and why. Particularly with pharmacological procedures, the details of the following should be given: drug formulation, dose, volume, concentration, site, route and frequency of administration, vehicle or carrier solution formulation and volume, and any evidence that the pharmacological agent used reaches the target tissue. With the surgical procedures, the surgical procedure description, anesthetic used, pre-and postsurgery analgesia, presurgery procedures (eg, fasting), aseptic techniques, monitoring, whether the procedure is terminal or not, duration of the procedure and the anesthesia, and physical variables measured should be reported. 65 A total of 31 (70.5%) of 44 studies used one of several approaches to elevate TGF-β1 levels in the TA (Table 1). Based on findings that the gene and protein expression of TGF-β1 were increased in the human PD plaque, 72 Lue and colleagues developed a rat model using an injection of cytomodulin, a TGF-β1-like peptide, into the TA. Six weeks following the injection, the TA of the rats showed a chronic cellular infiltration, elastosis, thickening of the TA, disorganization and clumping of collagen bundles, and expression of TGF-β1. 50,52 Later, Bivalacqua et al 48 reported that injection of the recombinant TGF-β1 protein produced similar effects but that a combined intervention of surgical trauma and TGF-β1 injection caused more profound PD-like changes. Other studies that used TGF-β1-elevating approach utilized adenovirus expressing TGF-β1. 36,40 Fibrin was the second most used inducer of fibrosis among the 44 studies that we identified. Five (11.4%) studies used fibrin alone or in combination with TGF-β1 or thrombin to induce fibrosis in TA. 17,30,42,44,54 Davila et al 44 was the first to study the use of fibrin injections into the TA to induce fibrotic plaques in rats. They suggested that in comparison with the TGF-β1 model, the fibrin model better resembled the pathophysiological event of a penile trauma (extravasation of fibrin), which is considered to be the etiological key event for human PD. In addition, the fibrin injection induced a faster plaque formation (2 weeks) and produced larger plaques than the TGF-β1 model. The authors found that the fibrin model reproduced histological features that correlate to human PD: collagen disorganization, fibrin deposition, elastin fragmentation, increased amounts of TGF-β1, inducible nitric oxide synthase, and reactive oxygen species. 42,44 Other fibrosis inducers were thrombin only 33 or in combination with fibrin, 17,30,54 allograft of TA tissue from another rat, 55 autologous blood 26 or plasma, 15 chlorhexidine, 23 sodium tetradecyl sulfate, 27,32 and extracorporeal shockwave (Table 1). 37 The only animal model that developed PD-like pathology spontaneously was Tsk mice. 38 The Tsk mouse model is characterized by dermal fibrosis and systemic sclerosis and has recently been shown to develop PD-like lesions. 38 These mice have a mutation in the gene encoding fibrillin-1, a large ECM structural protein and the major component of microfibrils and display hypodermal fibrosis and some other typical abnormalities of human systemic sclerosis. 73 The spontaneously occurring PD-like penile changes in Tsk mice include disorganization of the TA structure with accumulation of type I collagen, fibrous plaque formation, penile bending, and areas of chondroid metaplasia with heterotopic ossification. 38

Remarks
The analysis of the data has shown that most of the studies with TGF-β1 injection used 0.5 or 1 μg of the cytokine. There was 1 outlier study that used 50 μg. 14 We would suggest 0.5 to 1 μg in a small volume such as 50 to 100 μL using a small-gauge needle and microliter syringe. The vehicle in which TGF-β1 is dissolved should be used for the control group.
Although fibrin is the second most common inducer after TGF-β1, its poor solubility and high viscosity can be a technical challenge considering that the volume administered should be no more than 100 μL.
With tissue grafting and blood or plasma, the main issue is standardization of the inducing agents because tissue, blood, and plasma will have inherent variation in their cellular and protein content. This would be amplified in a small volume or space such as rodent TA.
With any of the new techniques, we suggest that the new model should be optimized and standardized in-house and should be reported accordingly. It should always be remembered that any handling, dissection, or surgery to the skin or to the TA is likely to trigger inflammatory response; therefore, appropriate sham control groups should be designed into the study.
Although we do welcome a spontaneous PD animal model, such as the Tsk mouse model, we also acknowledge that this model requires further characterization and optimization. Most importantly the erectile function in Tsk mice should be measured at different ages of the animal so that a correlation between fibrosis development and erectile dysfunction can be assessed.
Statement #6: We suggest that a detailed description of how fibrosis was measured should be given.

Remarks
The methods that were employed in measurement of fibrosis in the 44 publications that we have reviewed are listed in Table 2 in which we summarized the principles of the methods as well as their advantages and disadvantages.
Based on the previous assessment, we would suggest that Masson's trichome method should be utilized to measure the smooth muscle/fibrosis ratio. Any quantification from Masson's trichrome staining should be performed blindly (ie, by a person who is not aware which experimental group the samples belong to) in multiple regions of interest from multiple slides for each experimental group, ideally using an image analysis software. The researchers should also note that because this staining method is not protein specific, erythrocytes would for example be stained red like smooth muscle cells.
Our second suggestion would be to quantify the protein amount of ECMs and smooth muscle. Ideally this should be performed using Western blotting or enzyme-linked immunosorbent assay to measure relevant collagens (eg, collagen I and III), elastin, and α-smooth muscle actin. The source, catalog number and titers of the antibodies should be reported. The results should be normalized to an internal protein that is known not to be altered by fibrosis such as GAPDH. 74 Ratios between collagens/elastin and smooth muscle should be calculated and reported. We suggest Masson's trichrome staining and protein quantification as the minimum requirement for fibrosis evaluation. The other methods listed in Table 2 can also be employed depending on the research question and the project-specific endpoints. Quantitative PCR methods such as real-time quantitative PCR can be employed, as long as international MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines 75 are followed, to obtain quantitative data with low variability. Any results from PCR should be validated using protein expression since not all changes in messenger RNA expression necessarily translate to changes in protein expression. 76,77 Statement #7: A PD model does not need to cause penile curvature or calcification in rats.

Evidence
Of 44 publications we have identified, 5 measured penile curvature in rats. 15,18,23,40,55 Curvature was measured typically by measuring the angle of curvature in the photos with a protractor. Artificial erection using saline or water injection into the cavernous space was often employed before the angle was measured. Three studies assessed cartilage formation or calcification 36,40,55 using Safranin-O or Von Kossa staining.

Remarks
Rat penis shows distinct differences in anatomy and physiology compared with human penis. It has a cylindrical shaft and a bulbous glans with 90 • frontal flex. It has also a cylindrical bone (os penis) that extends from the distal end of the body to the tip of the glans. 78 Striated muscle contraction is required to straighten the glans during intromission. 79 Despite this uniquely different anatomy and physiology, rats are frequently used in research involving erectile function/dysfunction and PD due to the presence of human-like corpus cavernosum, TA, low cost, and relatively larger size than mice. For example, ICP measurements in rats in response to cavernous nerve stimulation have now been widely accepted standard model for erectile function assessment. 63 When a fibrosis inducing agent such as TGF-β1 or fibrin is injected to the shaft of the rat penis, a fibrotic plaque does indeed form. But it would be practically challenging to accurately measure the angle of any curvature formed by this plaque simply because of the anatomy of the rat penis. We suggest that such curvature measurements can be used to follow up the development of the plaque but should not be used as a primary end point. Similarly, fibrosis in rat TA does not always produce calcification and/or cartilage/bone formation. When it does, the amounts are too small to measure and quantify accurately. We suggest that the measures of calcification, cartilage/bone formation should not be used as primary end points. Statement #8: We suggest measuring erectile function using cavernous nerve electrostimulation in PD animal models.

Evidence
A total of 20 (45.5%) studies assessed erectile function either by measuring ICP in response to electrostimulation of the cavernous nerve (n = 19) or to papaverine injection (n = 1) ( Table 1). Particularly quantitative ICP: mean arterial pressure (MAP) ratios were successfully utilized to measure the effect and test hypotheses.

Remarks
Although not all patients with PD develop ED, erectile function measurement has proven to be a useful functional and quantitative proxy for PD in animal models. We suggest that ICP and MAP measurements should be performed under anesthesia; the ICP and MAP values and their ratios should be reported. Ideally representative tracings of ICP and MAP should also be presented. The erections should be induced by electrostimulation, rather than by vasoactive agents like papaverine, as fibrosis may alter the local pharmacokinetics of injected drugs. For other technical details of this procedure, the ESSM guidelines should be followed. 63 Statement #9: We suggest considering spontaneous recovery of fibrosis in the experimental design.

Evidence
Of 44 studies reviewed here, 29 (65.9%) tested a treatment. Of those 29 studies, 14 (48.3%) tested a treatment to prevent fibrosis meaning that the treatment was initiated on the day of or shortly after fibrosis induction (Table 1). Seven (24.1%) studies tested a treatment to reverse fibrosis, meaning that the treatment was initiated once the fibrosis was established (Table 1). Eight (27.6%) studies tested a treatment in both prevention and reversal mode, meaning that the treatment was initiated immediately after the induction of fibrosis in one group of animals and initiated once the fibrosis was established in another group of animals (Table 1). In reversal mode, the treatment was initiated on average ∼30 days after the fibrosis induction. The delay between the fibrosis induction and the start of the treatment varied largely between 15 and 45 days (Table 1).

Remarks
To test disease preventing agents, initiation of the treatment immediately or 1 to 2 days after the fibrosis induction (eg, TGF-β1 or fibrin) is highly suggested. The importance and necessity of a time-matched control group should be highlighted once again here because it would not be possible to draw any conclusion on the effectiveness of preventive treatment without such a control group.
The researchers should note that experimentally induced fibrosis in animal models may spontaneously resolve without any treatment as shown in bleomycin-induced pulmonary fibrosis, 80 systemic sclerosis 81 in mice, and TGF-β1-induced PD in rats. 21 It is therefore essential to have time-matched control groups. Pilot experiments should be performed to find the optimum delay time and the time for the study end; the results from these pilot studies should be reported.
A temporal analysis of Tsk model should be conducted to investigate whether fibrosis in TA of these mice undergo spontaneous resolution.
From the clinical translation point of view, the preventive treatment groups will be investigating the effect of treatment modalities targeting early-stage (acute phase) PD. The investigation into treatment modalities that target late-stage (chronic phase) PD is made difficult by the spontaneous resolution of fibrosis in animal models.

Conclusion
Several animal models for PD are available, but unfortunately none of the models mimic human PD precisely. The Sexual Medicine, 2023, Vol 11, Issue 4 13 animal models reviewed here are based on different approaches to induce PD-like changes, and each model may to some extent simulate 1 or more mechanisms or signs of the human pathology. To increase the validity and translatability of PD research in animal models, it is crucial that each of the models is methodologically standardized and optimized; doing so ensures that the models mimic the human physiology as closely as possible. In this document, we provided ESSM position statements for the correct and reproducible use of these animal models in PD research. We hope that the suggestions we made here will advance the PD field and will narrow the translational gap.